Many inherited metabolic diseases impair specialized epithelial cell function, but direct study of such cells is often impossible because they are inaccessible in the human. Nephropathic cystinosis is such a disorder, a prototypical lysosomal storage disease producing renal injury, with early onset of proximal tubular dysfunction and Fanconi's syndrome, and eventual renal failure by the second decade of life. The mechanisms of tubular dysfunction in this disease are not understood, largely because mutant tubular cells have not been available for study. Recently, we have established in vitro cultures of cystinotic renal tubular cells by harvesting cells from voided urine of cystinotic non-transplanted patients. These cells express differentiated features of renal proximal tubular cells in vitro, and express the storage defect, with cellular cystine levels up to 100-fold those in normal cultured renal proximal tubular cells. These cells can be depleted of their cystine stores by cysteamine, a free thiol compound which releases cystine from the lysosomes and is used clinically to treat these patients. In these studies, we propose to address the following hypotheses: 1) that the storage defect in these epithelial cells is due to a defective transporter for cystine egress from lysosomes, a defect which has not yet been established in epithelial cells in this disorder; 2) that transport defects in these cells are related to specific effects on polarized plasma membrane transport processes, and/or to nonspecific metabolic effects; 3) that Fanconi's syndrome which develops in these patients is directly related to elevated levels of intracellular cystine, and that depletion of intracellular cystine in these cells may normalize structural and functional abnormalities; and 4) that cystinotic tubular cells can be immortalized to provide large number of homogeneous optimally differentiated cells, which express the transport defect and can be used for future studies to attempt to define lysosomal transporter abnormalities. Non-depleted cystinotic cells will be compared with cystinotic cells depleted of cystine by cysteamine. Parallel studies will be performed in cultures of normal human proximal tubular cells, and in normal cells loaded with cystine using the soluble cystine compound cystine dimethyl ester as an additional in vitro model of the disease. In addition, we will use viral antigens to attempt to transform cystinotic renal tubular cells. Clones which are non-tumorigenic, and have epithelial morphology, monolayer growth pattern, express histochemical staining for renal proximal tubular brush border enzymes and other properties of non- transformed cystinotic cells as characterized will be selected for further propagation and characterization. Development of a cystinotic renal epithelial cell line would provide large numbers of homogeneous cells for study of the cellular pathophysiology of this disease, complementing and extending studies possible in non-transformed cultures of cystinotic cells.